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WO2013010333A1 - 反转录聚合酶链式反应的方法 - Google Patents

反转录聚合酶链式反应的方法 Download PDF

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Publication number
WO2013010333A1
WO2013010333A1 PCT/CN2011/077449 CN2011077449W WO2013010333A1 WO 2013010333 A1 WO2013010333 A1 WO 2013010333A1 CN 2011077449 W CN2011077449 W CN 2011077449W WO 2013010333 A1 WO2013010333 A1 WO 2013010333A1
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WO
WIPO (PCT)
Prior art keywords
reverse transcription
capillary
polymerase chain
chain reaction
reverse
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2011/077449
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English (en)
French (fr)
Inventor
苏城
邓秉华
李馥君
吴思颖
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Genereach Biotechnology Corp
Original Assignee
Genereach Biotechnology Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genereach Biotechnology Corp filed Critical Genereach Biotechnology Corp
Priority to CN201180071676.4A priority Critical patent/CN103687960A/zh
Priority to PCT/CN2011/077449 priority patent/WO2013010333A1/zh
Priority to US13/377,490 priority patent/US20130023010A1/en
Publication of WO2013010333A1 publication Critical patent/WO2013010333A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Definitions

  • the present invention relates to a method for biochemical reactions, and more particularly to a method for reverse transcription polymerase chain reaction by reacting a reverse transcription reaction with a polymerase chain
  • the reaction PCR
  • BACKGROUND OF THE INVENTION Reverse Transcription Polymerase Chain Reaction (referred to as Reverse Transcription Polymerase Chain Reaction)
  • RT-PCR is a reaction for detecting an RNA (ribonucleic acid) virus in which a messenger RNA (mRNA) is added to a reverse transcription reaction reagent (reverse transcriptase) to produce complementary DNA ( The complementary, cDNA), polymerase chain reaction, thereby amplifying the target DNA (deoxyribonucleic acid) sequence to millions of times for analysis.
  • mRNA messenger RNA
  • reverse transcriptase reverse transcriptase
  • a common reaction scheme for reverse transcriptase polymerase chain reaction is: first add a reverse transcription reaction reagent to a test tube, then add an RNA sample (analyte) for reverse transcription reaction, and then transcribe through a reverse transcription reaction.
  • the complementary DNA sample (analyte) is added to a PCR tube, and the enzyme and buffer required for the polymerase chain reaction are added for polymerase chain reaction.
  • the above-mentioned reverse transcription reaction and polymerase chain reaction of the sample and the reactants need to be added to the tubes of different reactions in batches.
  • the movement of the sample and the reactants in different tubes and the time of exposure to the air Long, the more likely it is to cause contamination of the sample and reactants, making the reaction results inaccurate.
  • the above reactants must be refrigerated at a low temperature to maintain the stability of the mass of the reactants and maintain the enzyme activity.
  • the transportation and storage methods that require cryogenic refrigeration will cause great inconvenience to transportation and storage, and the cost of cryogenic refrigeration equipment is high.
  • the main object of the present invention is to provide a method for reverse transcription polymerase chain reaction, wherein a reaction enzyme required for performing a reverse transcription reaction can be made into a reverse transcription lyophilization reagent in a capillary tube.
  • the RNA sample (analyte), buffer, polymerase and primer solution are added to the capillary during use, so that the reverse transcription reaction and the polymerase chain reaction do not need to be carried out in batches, and can be carried out in a capillary tube. .
  • Another object of the present invention is to use a pre-freeze drying program to remove more than 95-99% of water, so that the dried reverse transcription lyophilization reagent can be preserved for a long time without deterioration, and is completely dehydrated and light. Suitable for long-distance transportation and long-term storage at room temperature. Moreover, the reverse transcription lyophilization reagent can be stably stored at room temperature without affecting the experimental effect.
  • a further object of the present invention is to allow the reactants to be stored for a long period of time by a pre-freeze drying procedure, and because the reverse transcription lyophilization reagent is stored in a capillary tube, when reverse transcription polymerase chain reaction is required, The reverse lyophilization reagent is remelted (re-dissolved) in a capillary tube and directly reacted to shorten the operation time and avoid contamination caused by the movement of the reaction mixture.
  • the invention provides a method for reverse transcription polymerase chain reaction, the main feature is that a capillary tube is prepared, a reverse transcriptase required for a reverse transcription reaction is added into a capillary tube, and concentrated by vacuum to freeze the reverse transcription.
  • the dry reagent when used, the RNA sample, the buffer solution, the polymerase and the primer solution are added to the capillary, and mixed with the reverse transcription lyophilization reagent to be dissolved, so that the reverse transcription reaction and the polymerase chain reaction can be combined.
  • a method of reverse transcription polymerase chain reaction according to the present invention comprising the steps of: S1: preparing a capillary, and adding a reverse transcriptase to the capillary; S2: said to be placed in said capillary The reverse transcriptase is subjected to a pre-freeze drying procedure in which a reverse transcription lyophilization reagent is prepared.
  • the method of reverse transcription polymerase chain reaction according to the present invention further comprising the steps of: S3: adding a buffer and an RNA sample to the capillary to re-dissolve the reverse transcription lyophilization reagent.
  • S3 adding a buffer and an RNA sample to the capillary to re-dissolve the reverse transcription lyophilization reagent.
  • the method of reverse transcription polymerase chain reaction according to the present invention wherein in step S3, a polymerase is added to the capillary, and a primer solution is added to invert the RNA sample in the capillary After the reaction is recorded, the polymerase chain reaction can be carried out directly.
  • the method of reverse transcription polymerase chain reaction according to the present invention wherein in step S1, a polymerase is added to the capillary having the reverse transcriptase, and in step S2, the inversion is performed
  • the enzyme is subjected to the pre-freeze drying procedure in conjunction with the polymerase.
  • the method of reverse transcription polymerase chain reaction according to the present invention wherein the pre-freeze drying procedure comprises a low temperature freezing step and a drying step.
  • the method of reverse transcription polymerase chain reaction according to the present invention, wherein the cryogenic freezing step is to place the capillary in a freezer.
  • a method of reverse transcription polymerase chain reaction according to the present invention, wherein the low temperature freezing step is a The capillary is placed in the condenser.
  • the method of reverse transcription polymerase chain reaction according to the present invention wherein the drying step is a step of vacuum evacuating the capillary.
  • the method of reverse transcription polymerase chain reaction according to the present invention wherein a preheating procedure is performed after the pre-freeze drying procedure to maintain the activity of the reverse transcriptase in the reverse transcription lyophilization reagent.
  • a method of reverse transcription polymerase chain reaction according to the present invention wherein the preheating procedure is to use an increasing temperature gradient to warm the reverse lyophilization reagent for storage. Therefore, the present invention provides an improvement of the experimental steps, which not only ensures the accuracy of the reaction result, but also improves the convenience and efficiency of the experiment.
  • FIG. 1 is a flow chart of a method according to a first embodiment of the present invention.
  • Figure 2 is a diagram of the pre-freeze drying procedure of the present invention.
  • 3A to 3D are schematic views showing the operation of the method of the present invention.
  • Figure 4 shows the results of analysis of the product of the reverse transcription polymerase chain reaction by agarose gel electrophoresis.
  • Figure 5 is a flow chart of a method of a second embodiment of the method of the present invention.
  • Figure 6 is a flow chart of a method of a third embodiment of the method of the present invention.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to facilitate the description of the present invention, it will be described by way of specific embodiments. Various objects in the embodiments are depicted in terms of ratios, dimensions, amounts of deformation, or displacements that are suitable for illustration, and are not drawn to scale of actual components. Referring to FIG. 1, FIG. 2, FIG. 3A to FIG.
  • a method for reverse transcription polymerase chain reaction which comprises the steps S1 S2: Step S1: preparing a capillary tube 10, and a reverse transcriptase 11 (Fig. 3A) is added to the capillary 10 for performing a reverse transcription reaction. Step S2: performing a pre-freeze drying process 20 on the reverse transcriptase 11 placed in the capillary 10, the pre-freeze drying program 20 including a low-temperature freezing step 201 and a drying step 202, and the low-temperature freezing step 201 is to have a reverse transcriptase 11
  • the capillary 10 is placed in the freezer 21 (Fig.
  • the drying step 202 is a step of vacuum evacuating the capillary 10 having the reverse transcriptase 11, whereby the inversion can be made in the capillary 10.
  • the lyophilized reagent 111 is used for use in simultaneous reverse transcription reaction and polymerase chain reaction.
  • This embodiment may further include the step S3: adding a buffer (not shown) to the RNA sample 12 (as shown in FIG. 3C) in the capillary 10, so that the reverse transcription lyophilization reagent 111 is dissolved back, thereby making the reverse The transcription reaction and the polymerase chain reaction can be carried out together in the capillary 10.
  • the polymerase 13 can be added to the capillary 10 (as shown in FIG.
  • the program 20 includes a low temperature freezing step 201 and a drying step 202, and the low temperature freezing step 201 can be further divided into one freezing (sublimation freezing) and secondary freezing (desorption freezing), and the condition of one freezing is a capillary having a reverse transcriptase 11 10 is placed in the freezer 21, the temperature is controlled between -20 ° C and -40 ° C, and pre-frozen for 2-4 hours, in order to crystallize the reverse transcriptase 11 and sublimate excess water;
  • the freezing condition is that the reverse transcriptase 11 which is pre-frozen into crystal form is placed in a condenser (not shown), the temperature is controlled between -40 ° C and -60 ° C, and the condensation is 2-4 hours.
  • the purpose is to carry out preliminary drying. Thereafter, a drying step is carried out, and vacuum evacuation is carried out under a vacuum of less than 100 mTorr to complete the preparation of the reverse transcription lyophilization reaction agent 111.
  • Figure 4 is the result of reverse transcription polymerase chain reaction, in which the reverse transcription polymerase chain is carried out by a commonly used reverse transcription polymerase chain reaction reaction scheme and a reverse transcription lyophilization reagent. The reaction was used as a control.
  • the reverse transcription lyophilization reagent 111 is stored at 37 ° C for one week, and may be reconstituted by using a re-dissolving buffer, sterile water or a suitable diluent, and adding 2.5% glycerol as an expansion. Agent. Thereafter, the RNA sample 12, the polymerase 13 and the primer solution are added for reverse transcription polymerase chain reaction, and the two different reverse transcription polymerase chain reaction reactions of the present invention and the present invention are made by a black line in FIG. The interval, and the number 0-3 represents the result of obtaining samples from four individuals of water-supplying organisms suspected of being infected with the muscle necrosis virus.
  • S2 and S3 represent the standard plasmids of different amplification quantities, respectively, and n represents the normal water-producing nucleic acid.
  • M stands for molecular size marker.
  • the reverse transcription lyophilization reagent 111 can be directly dissolved in the capillary 10 and then directly reacted, thereby shortening the operation time and avoiding reverse transcription of the lyophilization reagent 111.
  • the pollution produced by the movement Referring to FIG.
  • a second embodiment of the present invention differs from the foregoing embodiment in that step S1A, step S2A and step S3A are performed, wherein step S1A: preparing the capillary 10 and adding reverse transcription to the capillary 10 Enzyme 11 and polymerase 13, compared to step S1, the difference is that the polymerase 13 is also added to the capillary 10 having the reverse transcriptase 11; Step S2A: is the reverse transcriptase to be placed in the capillary 10 11 pre-freeze drying process 20 with the polymerase 13 to obtain a reverse transcription lyophilization reagent 111, which is different from step S2 in that the reverse transcriptase 11 and the polymerase 13 are co-freeze-dried.
  • a third embodiment of the present invention differs from the first embodiment described above in that, in the present embodiment, step S2B is further performed: preheating the reverse transcription lyophilization reagent 111 That is, the pre-freeze drying program 20 may be followed by a preheating procedure to maintain the activity of the reverse transcriptase 11 in the reverse transcription lyophilization reagent 111, and the preheating program uses an increasing temperature gradient,
  • the condition is that the reverse transcription lyophilization reagent 111 is first stored at -10 ° C for six hours, and the reverse transcription lyophilization reagent 111 is stored at 26 ° C for six hours, and the reverse transcription is performed by using a stepwise temperature change.
  • the lyophilized reagent 111 can be stored at room temperature and sealed, and the reverse transcriptase 11 in the reverse lyophilization reaction agent 111 is prevented from being deactivated due to excessive temperature difference, so that the reverse transcriptase 11 remains active, which will be beneficial to improve The accuracy of the experimental results.
  • the method of the invention improves the cumbersomeness of the conventional reverse transcription polymerase chain reaction to be added to the reactants in batches, and also improves the inconvenience of the reactants to be transported and stored at low temperature, so that the reaction process is further improved. Convenience; summarize the features of the invention as follows:
  • the cleanliness of the operation in the capillary 10 is high, and the contamination of the bacteria and particles is reduced.
  • the reverse transcription polymerase chain reaction can be directly performed in the capillary 10 to avoid the enzyme and the enzyme.
  • the buffer is added in batches and the pollution generated by the movement is added successively. 2. Because the reverse transcription lyophilization reagent 111 is in the capillary 10, it can be directly re-dissolved and then subjected to reverse transcription polymerase chain reaction, which shortens the operation time and improves the efficiency of the experimental procedure.
  • the reverse transcription lyophilization reagent 111 stored in the capillary 10 can be stored for a long period of time without deterioration, and is completely dehydrated, light in weight, suitable for long-distance transportation at room temperature, and long-term storage. Moreover, the reverse transcription lyophilization reagent 111 can be stably stored at room temperature without affecting the experimental effect.
  • Reverse transcription lyophilization reagent 111 The treatment conditions are mild, and the crystal is dried at low temperature and low pressure; the decomposition and degeneration of reverse transcriptase 11 under high temperature and high pressure can be avoided.
  • Reverse transcription lyophilization reagent 111 has low water content, is not easy to be oxidized, is conducive to long-distance transportation and long-term preservation, and has good rehydration, and can be quickly hydrated and reduced to a state before lyophilization.

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Abstract

本发明提供一种反转录聚合酶链式反应的方法，其主要包括以下步骤：准备毛细管，将反转录反应所需的反转录酶加入毛细管中，经过预冻干燥程序，在该毛细管内制成反转录冻干反应剂，由此，在使用时可将RNA检体、缓冲液、聚合酶与引物溶液直接加入该毛细管中，与反转录冻干反应剂混合进行回溶，使反转录反应与聚合酶链式反应可一并于毛细管内进行，因而有助于提高实验流程的便利性。

Description

反转录聚合酶链式反应的方法 技术领域 本发明涉及一种生物化学反应的方法， 特别涉及一种反转录聚合酶链式反应的 方法，该方法通过使反转录反应与聚合酶链式反应 (PCR)在同一装置内进行，可提高 反应流程的便利性。 背景技术 反转录聚合酶链式反应 (Reverse Transcription Polymerase Chain Reaction, 简称
RT-PCR), 是用于检测 RNA (核糖核酸） 病毒的反应， 在该反应中， 将信使核糖核 酸（message RNA, mRNA)加入反转录反应试剂（反转录酶）以制造互补 DNA (complementary, cDNA), 再进行聚合酶链式反应， 从而将靶 DNA (脱氧核糖核酸） 序列 (target sequence)扩增至数百万倍， 用于进行分析。 反转录聚合酶链式反应常用的反应流程为： 先向试管中加入反转录反应试剂， 再加入 RNA检体 （分析物） 进行反转录反应， 之后再将经由反转录反应而转录出 的互补 DNA检体 （分析物） 加入 PCR试管中， 并加入聚合酶链式反应所需的酶与 缓冲液， 以进行聚合酶链式反应。 上述反转录反应与聚合酶链式反应的检体与反应 物需分批分次地添加至不同反应的试管内， 检体与反应物在不同试管中的移动与暴 露在空气中的时间越长， 越容易造成检体与反应物的污染， 使得反应结果失准。 并且， 上述反应物必须进行低温冷藏， 以维持反应物的质量的稳定与维持酶活 性。 需低温冷藏的运输与储存方式， 将对运输以及保存造成很大的不方便， 且低温 冷藏设备的成本较高， 同时， 若储存不当， 还可能造成反应物的变质， 或酶失去活 性， 进而影响反应结果的准确度。 发明内容 本发明的主要目的在于， 提供一种反转录聚合酶链式反应的方法， 其中， 可在 毛细管中将进行反转录反应所需的反应酶制成反转录冻干反应剂， 使用时将 RNA 检体（分析物）、 缓冲液、 聚合酶与引物溶液加入毛细管中， 由此使得反转录反应与 聚合酶链式反应无须分批分次进行， 可一并在毛细管中进行。 本发明的另一目的在于， 使用预冻干燥程序以去除 95-99%以上的水份， 使干燥 后的反转录冻干反应剂能长期保存而不致变质， 且因脱水彻底、 重量轻、 适合常温 下长途运输和长期保存。 且反转录冻干反应剂在室温下能稳定保存而不会影响其实 验效果。 本发明的又一目的在于， 使反应物经过预冻干燥程序而可保存较久， 且因为反 转录冻干反应剂储存于毛细管内， 在需进行反转录聚合酶链式反应时， 可在毛细管 内将反转录冻干反应剂回溶（复溶、 再溶解， re-dissolution)后直接进行反应， 缩短 操作时间并避免反应混合液移动所产生的污染。 本发明提供一种反转录聚合酶链式反应的方法， 主要特征在于， 准备毛细管， 将反转录反应所需的反转录酶加入毛细管中， 经过真空浓缩冷冻， 制成反转录冻干 反应剂， 使用时将 RNA检体、 缓冲液、 聚合酶与引物溶液加入毛细管中， 与反转 录冻干反应剂混合进行回溶， 使反转录反应与聚合酶链式反应可一并于毛细管内进 行。 根据本发明的反转录聚合酶链式反应的方法， 其中， 包括以下步骤： S1 : 准备 毛细管， 并向所述毛细管中加入反转录酶； S2: 对置于所述毛细管中的所述反转录 酶进行预冻干燥程序， 在所述毛细管内制成反转录冻干反应剂。 根据本发明的反转录聚合酶链式反应的方法， 其中， 还包括以下步骤： S3 : 向 所述毛细管中加入缓冲液与 RNA检体， 使所述反转录冻干反应剂回溶。 根据本发明的反转录聚合酶链式反应的方法， 其中， 于步骤 S3中， 将聚合酶加 入所述毛细管中， 并加入引物溶液， 使所述 RNA检体于所述毛细管中进行反转录 反应后， 可直接进行聚合酶链式反应。 根据本发明的反转录聚合酶链式反应的方法， 其中， 于步骤 S1中， 将聚合酶加 入具有所述反转录酶的所述毛细管中，并于步骤 S2中，使所述反转录酶与所述聚合 酶共同进行所述预冻干燥程序。 根据本发明的反转录聚合酶链式反应的方法， 其中， 所述预冻干燥程序包括低 温冷冻步骤与干燥步骤。 根据本发明的反转录聚合酶链式反应的方法， 其中， 所述低温冷冻步骤为将所 述毛细管置入冷冻库中。 根据本发明的反转录聚合酶链式反应的方法， 其中， 所述低温冷冻步骤为将所 述毛细管置入冷凝器中。 根据本发明的反转录聚合酶链式反应的方法， 其中， 所述干燥步骤为对所述毛 细管进行真空抽气的步骤。 根据本发明的反转录聚合酶链式反应的方法， 其中， 在所述预冻干燥程序之后 进行预热程序， 使所述反转录冻干反应剂中的反转录酶维持活性。 根据本发明的反转录聚合酶链式反应的方法， 其中， 所述预热程序为使用递增 的温度梯度， 使所述反转录冻干反应剂升温以便于储存。 因此， 本发明提供了实验步骤的改良， 除可确保反应结果的准确性， 还可提高 实验上的便利性与效率， 相比于现有技术， 本发明有助于提高实验流程的便利性， 并可避免检体与反应物在不同试管中移动而造成污染， 以及反应物的运输与保存不 当而造成反应物变质， 或酶失去活性。 附图说明 图 1为本发明第一种实施方式的方法流程图。 图 2为本发明的预冻干燥程序图。 图 3A〜图 3D为本发明方法的操作示意图。 图 4为反转录聚合酶链式反应的产物经琼脂糖凝胶电泳的分析结果。 图 5为本发明方法的第二种实施方式的方法流程图。 图 6为本发明方法的第三种实施方式的方法流程图。 具体实施方式 为便于说明本发明， 以具体实施例进行描述。 实施例中各种不同的对象按照适 于说明的比例、 尺寸、 变形量或位移量而描绘， 并非按实际元件的比例予以绘制。 请参照图 1、 图 2、 图 3A〜图 3D， 其为本发明的第一种实施方式， 其提供一种 反转录聚合酶链式反应的方法， 包括步骤 S1 S2: 步骤 S1 : 准备毛细管 10， 并向该毛细管 10中加入反转录酶 11(如图 3A)， 用于 进行反转录反应。 步骤 S2:对置于毛细管 10中的反转录酶 11进行预冻干燥程序 20，该预冻干燥 程序 20包括低温冷冻步骤 201与干燥步骤 202， 低温冷冻步骤 201为将具有反转录 酶 11的毛细管 10置入冷冻库 21中 (如图 3B)，而该干燥步骤 202为对具有反转录酶 11的毛细管 10进行真空抽气的步骤， 由此可在该毛细管 10中制成反转录冻干反应 剂 111， 用于在同时进行反转录反应与聚合酶链式反应时使用。 本实施例还可包括步骤 S3 : 在毛细管 10中加入缓冲液 (图中未示出)与 RNA检 体 12(如图 3C)， 使该反转录冻干反应剂 111回溶， 从而使反转录反应与聚合酶链式 反应可一并于毛细管 10中进行。 并且， 可于步骤 S3中， 将聚合酶 13加入该毛细管 10(如图 3D) (其可与 RNA检 体 12—同加入)， 并再加入引物溶液 (图中未示出）， 使 RNA检体 12于毛细管 10中 进行反转录反应后， 可直接进行聚合酶链式反应。 明了上述方法原理后， 以下针对本发明的运作及原理进行详细说明： 请一并参照图 2， 图 2为预冻干燥程序 20生产反转录冻干反应剂的主要流程， 其中， 预冻干燥程序 20包括低温冷冻步骤 201与干燥步骤 202， 低温冷冻步骤 201 又可分为一次冷冻 (升华冷冻)与二次冷冻 (解吸冷冻)，一次冷冻的条件为将盛有反转 录酶 11的毛细管 10置于冷冻库 21中， 温度控制在 -20°C至 -40°C之间， 预冻 2-4小 时， 目的在于使反转录酶 11形成结晶， 并使多余水分升华； 而二次冷冻的条件则为 将预冻成结晶状的反转录酶 11置入冷凝器 (图中未示出)中，温度控制在 -40°C至 -60°C 之间， 冷凝 2-4小时， 目的在于进行初步干燥。 之后进行干燥步骤， 在真空度低于 100毫托 （milliTorr) 的条件下进行真空抽气干燥， 从而完成反转录冻干反应剂 111 的制备。 请参照图 4， 其为反转录聚合酶链式反应的实验结果， 其中， 分别以常用的反 转录聚合酶链式反应流程以及使用反转录冻干反应剂进行反转录聚合酶链式反应作 为对照。 其中， 反转录冻干反应剂 111于 37°C情况下储存一星期， 需使用时可用回溶缓 冲液、 无菌水或适合稀释剂复原使其回溶， 并加入 2.5%的甘油作为膨胀剂。 之后， 加入 RNA检体 12、聚合酶 13与引物溶液进行反转录聚合酶链式反应， 图 4中以黑 线将常用的以及本发明的两种不同反转录聚合酶链式反应流程作出区隔， 而数字 0-3代表由 4个疑似传染性肌肉坏死病毒感染的水产生物个体取得样品的结果， S2 与 S3分别代表不同扩增数量的标准质粒 （plasmid), n代表正常水产生物核酸的阴 性对照， M代表分子大小标记。 由实验结果可看出， 常用的反转录聚合酶链式反应结果与本发明使用反转录冻 干反应剂 111进行反转录聚合酶链式反应的结果， 经琼脂糖凝胶电泳分析后没有太 大差异， 但本发明却能在实验过程中提高实验的便利性， 无论是针对反应物或缓冲 液的储存或运输， 且反转录冻干反应剂 111储存于毛细管 10 (见于图 3B) 内， 需进 行反转录聚合酶链式反应时，可在毛细管 10内将反转录冻干反应剂 111回溶后直接 进行反应， 缩短了操作时间并避免反转录冻干反应剂 111移动所产生的污染。 请参照图 5， 本发明的第二种实施方式， 与前述实施方式的差异在于进行步骤 S1A、 步骤 S2A与步骤 S3A， 其中步骤 S1A: 为准备毛细管 10， 并向该毛细管 10 中加入反转录酶 11与聚合酶 13， 与步骤 S1相比， 差异为还将该聚合酶 13加入具 有该反转录酶 11 的该毛细管 10中； 步骤 S2A: 为将置于该毛细管 10的反转录酶 11与该聚合酶 13共同进行预冻干燥程序 20， 得到反转录冻干反应剂 111， 与步骤 S2相比，差异为使该反转录酶 11与该聚合酶 13共同进行该预冻干燥程序 20; 步骤 S3A: 为向该毛细管 10中加入缓冲液、 RNA检体 12与引物溶液， 将该反转录冻干 反应剂 111回溶， 即可使反转录反应与聚合酶链式反应一并于毛细管 10中进行， 由 此将更便利于实验流程的进行。 最后请参照图 6， 本发明的第三种实施方式， 与前述的第一种实施方式的差异 在于， 本实施方式中还进行步骤 S2B: 对该反转录冻干反应剂 111进行预热程序， 即， 该预冻干燥程序 20之后还可进行预热程序， 使该反转录冻干反应剂 111中的反 转录酶 11维持活性， 而该预热程序为使用递增的温度梯度， 其条件为先将反转录冻 干反应剂 111以 -10°C储存六小时， 再将反转录冻干反应剂 111以 26°C储存六小时， 利用阶段性的温度改变， 使反转录冻干反应剂 111可常温密封保存， 避免反转录冻 干反应剂 111中的反转录酶 11因温差过大导致酶失去作用， 使反转录酶 11保持作 用活性， 如此将有利于提高实验结果的准确性。 综上所述， 本发明的方法改善了常用反转录聚合酶链式反应需分批逐次加入反 应物的繁琐， 也改善了反应物需低温冷藏运输与储存的不便之处， 使反应流程更加 便利； 总结本发明的特点如下：
1. 在毛细管 10 内操作的洁净度高， 减少杂菌和微粒的污染， 制成反转录冻干 反应剂 111后可直接于毛细管 10中进行反转录聚合酶链式反应，避免酶与缓冲液分 批逐次添加与移动所产生的污染。 2. 因反转录冻干反应剂 111在毛细管 10内， 可直接回溶后进行反转录聚合酶 链式反应， 缩短操作时间， 提高实验流程效率。
3. 储存于毛细管 10中的反转录冻干反应剂 111能长期保存而不致变质， 且因 脱水彻底、 重量轻、 适合常温下长途运输和长期保存。 且反转录冻干反应剂 111在 室温下能稳定保存而不会影响其实验效果。
4. 反转录冻干反应剂 111处理条件温和， 在低温低压下结晶干燥； 可避免高温 高压下反转录酶 11的分解变性。
5. 反转录冻干反应剂 111含水量低，不易被氧化，有利于长途运输和长期保存， 且复水性好， 能迅速吸水还原成冻干前状态。

Claims

权 利 要 求 书
1. 一种反转录聚合酶链式反应的方法， 其特征在于， 包括以下步骤：
S1 : 准备毛细管 （10)， 并向所述毛细管 （10) 中加入反转录酶 （11 );
S2: 对置于所述毛细管 （10) 中的所述反转录酶 （11 ) 进行预冻干燥程序 (20)， 在所述毛细管 （10) 内制成反转录冻干反应剂 （111 )。
2. 根据权利要求 1所述的反转录聚合酶链式反应的方法， 其特征在于， 还包括以 下步骤：
S3： 向所述毛细管 （10) 中加入缓冲液与 RNA检体 （12)， 使所述反转录 冻干反应剂 （111 ) 回溶。
3. 根据权利要求 2所述的反转录聚合酶链式反应的方法， 其特征在于， 于步骤 S3 中， 将聚合酶 （13 )加入所述毛细管 （10) 中， 并加入引物溶液， 使所述 RNA 检体 （12) 于所述毛细管 （10) 中进行反转录反应后， 可直接进行聚合酶链式 反应。
4. 根据权利要求 1所述的反转录聚合酶链式反应的方法， 其特征在于， 于步骤 S1 中， 将聚合酶 （13 ) 加入具有所述反转录酶 （11 ) 的所述毛细管 （10) 中， 并 于步骤 S2中， 使所述反转录酶（11 )与所述聚合酶（13 )共同进行所述预冻干 燥程序 （20)。
5. 根据权利要求 1所述的反转录聚合酶链式反应的方法， 其特征在于， 所述预冻 干燥程序 （20) 包括低温冷冻步骤 （201 ) 与干燥步骤 （202)。
6. 根据权利要求 5所述的反转录聚合酶链式反应的方法， 其特征在于， 所述低温 冷冻步骤 （201 ) 为将所述毛细管 （10) 置入冷冻库 （21 ) 中。
7. 根据权利要求 5所述的反转录聚合酶链式反应的方法， 其特征在于， 所述低温 冷冻步骤 （201 ) 为将所述毛细管 （10) 置入冷凝器中。
8. 根据权利要求 5所述的反转录聚合酶链式反应的方法， 其特征在于， 所述干燥 步骤 （202) 为对所述毛细管 （10) 进行真空抽气的步骤。
9. 根据权利要求 1所述的反转录聚合酶链式反应的方法， 其特征在于， 在所述预 冻干燥程序（20)之后进行预热程序， 使所述反转录冻干反应剂（111 ) 中的反 转录酶 （11 ) 维持活性。
10. 根据权利要求 9所述的反转录聚合酶链式反应的方法， 其特征在于， 所述预热 程序为使用递增的温度梯度，使所述反转录冻干反应剂（111 )升温以便于储存。
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